Phase 3 cultures of human diploid fibroblasts are considered to represent a cellular model reflecting organismal senescence. While it is known that a large fraction of the cells in phase 3 cultures are nonproliferating cells, we know relatively little about the metabolic changes which characterized this end-state cell and, most important, the significance of such changes to the biologic process known as senescence. Studies in our laboratory over the past 5 years have suggested a new model for regulating protein mass: (a) a stochastic cell cycle in which basal protein degradation is randomly activated in the cell, thereby rapidly reducing cytoplasm mass; (b) return of the cell to an inactivate degradative state in which the cytoplasm mass is renewed; and (c) the intersection of this protein-regulating cycle with the proliferative cycle (controlling DNA mass). This model suggests that the pathology of cellular senescence may reflect a failure of this protein-renewal cycle; preliminary experiments in our laboratory suggest that a large fraction of the cells in phase 3 cultures have left the protein-regulating cycle. The proposed experiments designed to critically examine the hypothesis that phase 3 cultures are comprised of two subpopulations with respect to metabolism of the slow-turnover protein pool -- a subpopulation showing normal kinetics and a larger subpopulation of out-of-cycle cells which are not renewing the sto protein pool. Four approaches will be used: (1) studies involving both labeling and decay of label in the sto proteins of human fibroblasts in phase 3 cultures; (2) autoradiographic studies on the labeling of the sto protein pool in these cultures; (3) similar studies on subpopulations of small and large cells separated by cell sorting; (4) ultrastructural studies on these cell populations relating biochemical and structural features in these cell subpopulations. These experiments should help to define both a new cell cycle, likely to be important both in the renewal of the cytoplasmic mass and in growth regultion, and the possible alterations in this cycle which may be fundamental to our understanding of the cellular basis of senescence. Finally, the consequences of a failure of this renewal cycle are likely to be significant in terms of progressive cell dysfunction and cellular organelle pathology.